Surfactant Protein B (SP-B) is important for the normal functioning of lung surfactant. Using the known three-dimensional structures of proteins which also belong to the "saposin" class, early SP-B models predicted that the disulfide cross-linked, N- and C-terminal domains fold as charged amphipathic helices, and further that these adjacent helices may participate in critical surfactant activities. On a small-scale, we have chemically synthesized a 34 residue, disulfide-linked N-terminal - C-terminal peptide construct (i.e., "Mini-B") that emulates the in vitro - in vivo structure and function of the 79 residue parent protein in lung surfactant lipids. However, scale-up of the synthesis of Mini-B by either chemical synthetic methods or through a bacterial expression system has proven to be problematic. In this SBIR grant application, we propose to address this issue by utilizing a cell-free expression system (CFES) that would facilitate the large-scale production of high-purity Mini-B peptide. Development of a commercially viable production method will permit the use of the Mini-B peptide in biomedical applications, including lung surfactant replacement therapies such as Neonatal Respiratory Distress Syndrome (NRDS), Adult Respiratory Distress Syndrome (ARDS), as well as facilitate the development of novel delivery systems to the lung for antibiotics, anti-inflammatory agents, and genetic material. [unreadable] [unreadable]